Biological substance detection method using florescent dye-containing particle

ABSTRACT

A biological substance detection method for detecting a biological substance specifically in a pathological specimen, comprising a step of immunologically staining the pathological specimen using a fluorescent label, a step of staining the pathological specimen with a staining reagent for morphology observation purposes (eosin) to observe the morphology of the pathological specimen, a step of irradiating the stained pathological specimen with excited light to cause the emission of a fluorescent and detecting the biological substance in the pathological specimen. In the step of immunologically staining the pathological specimen, a special fluorescent particle for which the excitation wavelength appears in a region that is different from the excitation wavelength region of eosin is used as the fluorescent label.

CROSS REFERENCE TO RELATED APPLICATION

This Application is a 371 of PCT/JP2011/069553 filed on Aug. 30, 2011,which claimed the priority of Japanese Patent Application No.2010-193154 filed on Aug. 31, 2010, both applications are incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a biological substance detectionmethod, and specifically relates to tissue staining for multiplystaining a tissue with fluorescent labels.

BACKGROUND ART

As one of medical diagnoses, a pathological diagnosis is performed. Apathologist diagnoses a disease using a tissue section taken from ahuman body and notifies a clinician of the necessity or unnecessity of atherapy or operation. On the basis of conditions of a patient andpathological diagnosis, a physician determines a drug treatment plan,and a surgeon determines whether or not an operation should beperformed.

In pathological diagnosis, it is widely performed to prepare a tissuesample by slicing a tissue specimen obtained through resection of anorgan or needle biopsy into a thickness of about several micrometers andobserve an enlarged image of the tissue sample with an opticalmicroscope for obtaining various findings. In many oases, the sample isprepared by fixing a taken tissue through dehydration and paraffinblocking, slicing it into a thickness of several micrometers, andremoving the paraffin. Since the sample, which barely absorbs andscatters light, is substantially colorless and transparent, stainingwith a dye is usually performed prior to the observation.

As a staining method, various staining methods have been proposed.

In particular for the tissue sample, hematoxylin-eosin staining (HEstaining) using two dyes, hematoxylin and eosin, is typically used as amorphological observation staining for observing the morphology of thesample (Non-Patent Document 1 and Patent Documents 1 and 2).

The hematoxylin stains cell nuclei, calcareous sites, cartilage tissue,bacteria, and mucus to a color of from indigo blue to light blue. Theeosin stains cytoplasm, interstitial tissue, various fibers,erythrocytes, and keratinocytes to a color of from red to deep red. Apathologist makes a diagnosis on the basis of morphological informationand staining information such as changes in size and shape of cellnuclei and changes in tissue pattern in the microscopic image of thestained tissue sample.

As other staining for morphological observation, for example,Papanicolaou staining (Pap staining) used for cytodiagnosis is alsoknown.

In pathological diagnosis, immunological observation calledimmunostaining where a molecular target is stained for confirming theexpression of molecular information of a sample in order to diagnosedysfunction such as abnormal expression of a gene or a protein isperformed.

The immunostaining, for example, employs dye staining with an enzyme(DAB staining). The DAB staining stains antigens as observation objectswith antibodies modified so as to be stained by a dye, and measures theantigen level through observation. Alternatively, fluorescent labelingis employed. The fluorescent labeling stains antigens as objects withantibodies modified with a fluorescent dye and measures the antigenlevel through observation.

An attempt has currently been made to simultaneously performmorphological and immunological observations of a sample. For example,it has teen tried to perform HE staining for morphological observationand DAB staining for immunological observation simultaneously (PatentDocument 3).

However, the staining with an enzyme label such as DAB staining developsa color similar to the color in HE staining to preclude the distinctionbetween the staining by an enzyme label and the HE staining, which makesthe simultaneous observation difficult. In addition, in DAB staining,the concentration of the stain considerably varies depending onenvironmental conditions such as temperature and time to preclude theestimation of the actual amount of, for example, antibodies on the basisof the concentration of the stain.

Meanwhile, a fluorescent label is used in pathological diagnosis.

The fluorescence method has superior quantitative characteristicscompared with DAB staining (Non-Patent Document 1).

However, simultaneous performance of pathological diagnosis andmorphological observation with fluorescent labels has a disadvantage inthat results of the staining are readily affected by the fluorescence ofthe staining reagent used for the tissue staining. A possiblecountermeasure is to use an infrared excitation/emission fluorescent dyewhich is not affected by visible light (Patent Document 4). For example,infrared-emitting dyes such as Alexa Fluor 647 (Molecular Probes) andCY5 (GE Healthcare) are known.

PRIOR ART DOCUMENTS Patent Documents

[Patent Document 1] PCT Japanese Translation Patent Publication No.2001-525580

[Patent Document 2] Japanese Patent Laid-Open No. 2009-115599

[Patent Document 3] Japanese Patent Laid-Open No. 2010-134195

[Patent Document 4] International Publication No. WO 2000/006006

Non-Patent Document

[Non-Patent Document 1] “Shindan ni yakudatsu men-eki soshiki shindan(Immuohistochemistry useful for diagnosis)”, Bunkodo

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

Currently, a fluorescent dye is used in the immunofluorescent staining,which causes a problem that fluorescence intensity is low. In addition,in a case of using an infrared excitation/emission dye, an emissionwavelength thereof is outside the visible light region. Thus,confirmation by visual observation is difficult, and an expensivedetection device is required. Accordingly, the observation needsexpensive instruments such as a confocal laser microscope.

Given the above, a main object of the present invention is to provide amethod of detecting a biological substance, and the method is capable ofdiscriminating between staining for morphological observation andimmunostaining using fluorescent labels in simultaneous staining formorphological and immunological observation, without any infraredexcitation/emission fluorescent dye.

Means for Solving the Problem

According to a first aspect of the present invention for solving theabove-described problems, there is provided a biological substancedetection method of specifically detecting a biological substance in apathological section, and the biological substance detection methodincludes

immunostaining the pathological section with a fluorescent label;staining the pathological section with a staining reagent formorphological observation; and detecting the biological substance in thepathological section through fluorescence emission by irradiation of thestained pathological section with excitation light, and

in the immunostaining of the pathological section, a fluorescentnanoparticle including phosphor or a semiconductor and having anexcitation wavelength region different from an excitation wavelengthregion of the staining reagent is used as the fluorescent label.

According to a second aspect of the present invention, there is provideda biological substance detection method of specifically detecting aecological substance in a pathological section, and the biologicalsubstance detection method includes

immunostaining the pathological section with a fluorescent label;staining the pathological section with a staining reagent formorphological observation; and

detecting the biological substance in the pathological section throughfluorescence emission by irradiation of the stained pathological sectionwith excitation light, and

in the immunostaining of the pathological section, a fluorescentdye-containing particle including an organic or inorganic materialparticle containing a fluorescent dye and having an excitationwavelength region different from an excitation wavelength region of thestaining reagent is used as the fluorescent label.

According to a third aspect of the present invention, there is provideda biological substance detection method of specifically detecting abiological substance in a pathological section, and the biologicalsubstance detection method includes

immunostaining the pathological section with a fluorescent label,staining the pathological section with a staining reagent formorphological observation; and

detecting the biological substance in the pathological section throughfluorescence emission by irradiation of the stained pathological sectionwith excitation light, and

in the immunostaining of the pathological section, a fluorescentnanoparticle-containing particle including an organic or inorganicmaterial particle containing a fluorescent nanoparticle includingphosphor or a semiconductor and having an excitation wavelength regiondifferent from an excitation wavelength region of the staining reagentis used as the fluorescent label.

Effects of the Invention

According to the present invention, in simultaneous staining formorphological and immunological observation, the method can discriminatebetween staining for morphological observation and immunostaining usingfluorescent labels, without any infrared excitation/emission fluorescentdye.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 This is a diagram schematically illustrating a fluorescencespectrum and an excitation spectrum of eosin.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments for implementing the present invention will bedescribed. However, the present invention should not be limited thereto.

The method of detecting a biological substance according to a preferredembodiment of the present invention specifically detects a biologicalsubstance in a pathological section and basically includes step (1) ofimmunostaining the pathological section with a fluorescent label, step(2) of staining the pathological section with a staining reagent formorphological observation, and step (3) of detecting the biologicalsubstance in the pathological section through fluorescence emission byirradiation of the stained pathological section with excitation light.

In particular, in the step (1) of immunostaining the pathologicalsection a specific fluorescent nanoparticle, a fluorescentdye-containing particle, or a fluorescent nanoparticle-containingparticle having an excitation wavelength region different from theexcitation wavelength region of the staining reagent for morphologicalobservation is used as the fluorescent label.

The details of characteristics and types of the fluorescent label, theimmunostaining, the staining for morphological observation and the likeare as follows.

Here, either of the step (1) of immunostaining or the step (2) ofstaining for morphological observation may be performed first, that, is,the order (former and latter) is not limited.

[Fluorescent Label]

Eosin used in HE staining emits fluorescence under certain conditionsfor microscopic observation. The absorption wavelength region of eosinoverlaps the excitation wavelength regions of many fluorescent labels.Thus, there has been a problem that the light emission of eosin used forstaining interferes with the observation, of a fluorescent label. FIG. 1shows a fluorescence spectrum (excitation wavelength: 520 nm) and anexcitation spectrum (fluorescence wavelength; 540 nm) of eosin. Theexcitation spectrum shows that eosin is efficiently excited in awavelength region of less than 350 nm and in a wavelength region ofhigher than 450 nm and less than 550 nm.

Thus, it is required that the fluorescent label according to theembodiment is excited in a wavelength region other than the abovewavelength regions, i.e., in a wavelength region of 350 to 450 nm or ina longer wavelength region of 550 nm or more.

The emission wavelength of the fluorescent label according to theembodiment must be in the longer wavelength side of 550 nm or more inviews of absorption and emission of eosin and tissue autofluorescence,and further, is preferably 700 nm or less in terms of necessity ofvisual confirmation in fluorescence microscopic observation. Inparticular, in terms of visual sensitivity, the emission wavelength ofthe fluorescent label according to the embodiment is preferably from 590to 650 nm (590 nm or more and 650 nm or less) and more preferably from590 to 630 nm (590 nm or more and 630 nm or less.

[Type of Fluorescent Label]

A fluorescent label having a higher brightness is preferred in terms ofthe signal ratio to the fluorescence of eosin and a cellularauto-fluorescence as noise. Thus, as the fluorescent label of thepresent invention, a fluorescent nanoparticle (A), a fluorescentdye-containing particle (B), or a fluorescent nanoparticle-containingparticle (C) that has a high brightness level compared, with brightnessof a fluorescent dye is suitably used.

[(A) Fluorescent Nanoparticles]

The fluorescent nanoparticles used in the present invention have aparticle size of 1 to 500 nm, preferably 10 to 200 nm.

The fluorescent nanoparticle is composed of a semiconductor or phosphor.

As for the semiconductor, a group I-VI semiconductor such as ZnSe, ZnTe,CdSe, CdTe, PbS, PbSe, or PbTe, or a group II-VI semiconductor such asAlAs, AlSb, GaP, GaAs, GaSb, InP, InAs, or InSb can be used. In terms oftoxicity, GaP and InP are suitable used.

The Phosphor can be an Oxide Phosphor.

The phosphor is composed of, for example, a matrix of such as Y₂O₃,YVO₄, ZnO, or ZnS and an emission center of such as Eu or Nd.

(Paragraphs 1-3: Omitted) . . .

As for the fluorescent dye to be contained, rhodamine dye molecules,Aleza Fluor (manufactured by Invitrogen Corporation) dye molecules,BODIPY (manufactured by Invitrogen Corporation) dye molecules, Texas Reddye molecules, oxazine dye molecules, aromatic ring dye molecules, andcarbopyronine dye molecules can be given as examples.

Specific examples of the dye can be such as 5-carboxy-rhodamine,6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G,tetramethylrhodamine, and X-rhodamine; Alexa Fluor 555, Alexa Fluor 568,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635,Alexa Fluor 4647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700,Alexa Fluor 750, BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, and BODIPY 650/665 (these are manufactured by InvitrogenCorporation); Cy5, Cy5.5,1,3-bis[4-(dimethylamino)-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediyliumdihydroxide, bis(inner salt),1,3-bis[4-(dimethylamino)phenyl]-2,4-dihydroxycyclobutenediyliumdihydroxide, bis(inner salt), . . . .

[(C) Fluorescent Nanoparticle-Containing Particles]

The fluorescent nanoparticle-containing particles used, in the presentinvention are organic or inorganic particles containing the fluorescentnanoparticles described in the above (A).

The fluorescent nanoparticles may be introduced into the particles byany method. For example, the particles may be synthesized by bindingfluorescent nanoparticles to a monomer as a raw material of theparticles, or fluorescent nanoparticles may be introduced into particlesby adsorption.

The excitation wavelength can be adjusted to be suitable for observationby controlling the particle size, matrix composition, and impurity levelof the fluorescent nanoparticles to be contained.

It is required that the fluorescent nanoparticles are excited in thewavelength region of 350 to 450 nm or in a longer wavelength region of550 nm or more, so as not to overlap the absorption wavelength region ofeosin. The types of phosphor described in the above section regardingthe fluorescent nanoparticles are given as examples.

The size of the particles containing the fluorescent nanoparticlesranges from 10 to 500 nm, and preferably from 50 to 200 nm.

[Staining for Morphological Observation]

In the staining for morphological observation, particularly regardingthe staining for morphological observation of tissue samples,hematoxylin-eosin staining (HE staining) using two dyes, hematoxylin andeosin, is typically used in the staining for morphological observationof the morphology of a sample. The staining for morphologicalobservation is not limited thereto, and examples of other staining formorphological observation is such as Papanicolaou staining (Papstaining) used for cytodiagnosis.

In the HE staining, the hematoxylin stains cell nuclei, calcareoussites, cartilage tissue, bacteria, and mucus to a color of from indigoblue to light blue; and the eosin stains cytoplasm, interstitial tissue,various fibers, erythrocytes, and keratinocytes to a color of from redto deep red. However, the staining for morphological observation is notlimited thereto. Cell nuclei, calcareous sites, cartilage tissue,bacteria, and mucus may be stained by a hematoxylin analog or a dyehaving an absorption wavelength similar to an absorption wavelength ofhematoxylin to a color of from indigo blue to light blue; and cytoplasm,interstitial, tissue, various fibers, erythrocytes, and heratinocytesmay be stained, by an eosin analog or a dye having an absorptionwavelength similar to an absorption wavelength of eosin to a color offrom red to deep red.

[Immunostaining]

As a method of immunostaining of a tissue, fluorescent staining issuitable used.

The fluorescent staining is a method of staining an antigen site with afluorescent label. As a method other than the fluorescent staining, forexample, dye staining with an enzyme (DAB staining) is known. However,this method, is inferior to the fluorescent staining in sensitivity.

Staining can be performed by, for example, a method of staining anantigen with a label prepared by directly binding a fluorescent label toprimary antibodies (primary antibody method); a method of staining anantigen to which a primary antibody is bound with a label prepared bydirectly binding a fluorescent label to secondary antibodies (secondaryantibody method); or a method of staining an antigen to which primaryantibodies are bound and secondary antibodies modified by avidin orstreptavidin with a label prepared by directly binding a fluorescentlabel to biotin (biotin-avidin method or sandwich method).

A Primary antibody used in staining may be any antibody and is selecteddepending on the target to be subjected to immunostaining of a tissue.For example, staining of an HER2 antigen uses an anti-HER2 antibody. Asecondary antibody may be any antibody and is selected depending on theprimary antibody. For example, anti-mouse, rabbit, bovine, goat, sheep,dog, and chicken antibodies can be given as examples.

The fluorescent label may be bound to an antibody or blot in by anyknown method, for example, amidation of carboxylic acid with amide,sulfidation of maleimide with, thiol, imination of aldehyde with amine,or amination of epoxy with amine.

Although tissue staining has been described above, the present inventionis not limited thereto and can also be applied to cell staining.

Examples

Hereinafter, the present, invention will be described in detail byExamples, but is not limited thereto.

[Preparation of Samples]

(Sample 1: Fluorescent Nanoparticles)

CdSe/ZnS fluorescent nanoparticles (Qdot655, Invitrogen Corporation)modified with PEG having amino groups at the ends were prepared asfluorescent nanoparticles for antibody binding.

Meanwhile, anti-human ER antibodies were subjected to reductiontreatment with 1M dithiothreitol (DTT), and excess DTT was removed by agel filtration column to obtain a solution of the reduced antibodiescapable of bonding so silica particles.

The fluorescent nanoparticles for antibody binding and the reducedantibodies were mixed in PBS containing 2 mM EDTA, followed by reactionfor 1 hour. The reaction was stopped by adding 10 mM mercaptoetanol. Theobtained solution was concentrated with a centrifugal filter, andsubsequently, the unreacted antibodies and the like were removed by agel filtration column for purification to obtain fluorescentnanoparticles to which the anti-human ER antibodies were bound.

(Sample 2: Fluorescent Nanoparticles)

The same fluorescent nanoparticles to which the anti-human ER antibodieswere bound as sample 1 were prepared.

In the observation described below, sample 1 was observed at anexcitation wavelength of 375 nm, and sample 5 was observed at anexcitation wavelength of 575 nm. That is, the same fluorescentnanoparticles prepared as samples 1 and 2 were observed at differentexcitation wavelengths.

(Sample 3: Fluorescent Nanoparticles)

CdSe/ZnS fluorescent nanoparticles (Qdot605, Invitrogen Corporation)modified with PEG having amino groups at the ends were used asfluorescent nanoparticles for antibody binding.

Fluorescent nanoparticles to which the anti-human ER antibodies werebound were prepared as sample 1 with the exception described above.

(Sample 4: Fluorescent Dye-Containing Particles)

An organoalkoxysilane compound was obtained by mixing 9.9 rag of thefluorescent dye, CY5-SE (manufactured by Roche) and 3 μL or3-aminopropyltrimethoxysilane (KBM903, manufactured by Shin-EtsuSilicone) in DMP. The obtained organoalkoxysilane compound in an amountof 0.6 ml, was mixed with 48 ml, of ethanol, 0.6 mL of tetraethoxysilane(TEOS), 2 mL of water, and 2 mL of 28% aqueous ammonia for 3 hours. Theliquid mixture prepared in the above was centrifuged at 10000 G for 20minutes, and the supernatant was removed. Thereafter, ethanol was addedto disperse the precipitate therein, and the solution was centrifugedagain. The precipitate was washed twice with each of ethanol and purewater by the same procedure. The obtainedtetramethylrhodamine-containing silica nanoparticles were observed withan SEM. The average particle diameter was 104 nm, and the coefficient ofvariation was 12%.

The obtained phosphor-containing silica nanoparticles were adjusted to a3 nM concentration with a phosphate buffer physiological saline solution(PBS) containing 2 mM ethylenediaminetetraacetric acid (EDTA). Thissolution was mixed with SM(PEG)12(succinimidyl-[N-maleimidopropionaido)-dodecaethyleneglycol] ester,manufactured by Thermo Scientific K.K.) in a final concentration of 10mM, followed by reaction for 1 hour. The solution mixture wascentrifuged at 10000 G for 20 minutes. The supernatant was removed.Thereafter, PBS containing 2 mM EDTA was added to disperse theprecipitate therein. The solution was centrifuged again. The precipitatewas washed three times by the same procedure to obtain fluorescentdye-containing particles for antibody binding.

Meanwhile, anti-human ER antibodies were subjected to reductiontreatment with 1M dithiothreitol (DTT), and excess DTT was removed by agel filtration column to obtain a solution of the reduced antibodiescapable of bonding to silica particles.

The fluorescent dye-containing particles for antibody binding and thereduced antibodies prepared above were mixed in PBS containing 2 mMEDTA, followed by reaction for 1 hour. The reaction was stopped byadding 10 mM mercaptoethanol. The obtained solution was centrifuged at10000 G for 20 minutes, and the supernatant was removed. Thereafter, PBScontaining 2 mM EDTA was added to disperse the precipitate therein. Thesolution was centrifuged again. The precipitate was washed three timesby the same procedure to obtain fluorescent dye-containing particles towhich the anti-human ER antibodies were bound.

(Sample 5-1: Fluorescent Dye-Containing Particles)

TAMRA dye (manufactured by FCC) was used as a fluorescent dye.

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized, as sample 4 with the exception describedabove.

(Sample 5-2: Fluorescent Dye-Containing Particles)

Texas Red dye (manufactured by Sigma-Aldrich Co.) was used as afluorescent dye.

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized as sample 4 with the exception describedabove.

(Sample 5-3: Fluorescent Dye-Containing Particles)

Oxazine 170 dye (manufactured by Sigma-Aldrion Co.) was used instead ofthe fluorescent dye in the preparation of sample 4, and3-glycidyloxypropyltrimethoxysilane (manufactured by TCI CO., LTD.) wasused instead of 3-aminopropyltrimetoxysilane (KBM903, manufactured byShin-Etsu Silicone).

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized as sample 4 with the exceptions describedabove.

(Sample 5-4: Fluorescent Dye-Containing Particles)

First, 2.5 mg of Oxazine 170 (manufactured by Sigma-Aldrich Co.) wasadded to 22.5 mL, of water, and subsequently placed on a hot stirrer andwas heated at 60° C. for 20 minutes. Then, 1.5 g of Nikalac MX-035(manufactured by Nippon Carbide Industries Co., Inc.) was added thereto,followed by heating with stirring for further 5 minutes.

Subsequently, 100 μL of formic acid was added to the above solution,followed by heating with stirring at 60° C. for 20 minutes, and then thesolution was cooled at room temperature.

After cooling, the reaction mixture was put in a centrifuge tube, andthe tube was set to a centrifugal separator, followed by centrifligationat 12000 rpm for 20 minutes. The supernatant was removed.

Subsequently, the reaction mixture after the supernatant was removed waswashed with ethanol and water.

The obtained particles were modified with anti-human ER antibodies usingSM(PEG)12 (succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester, manufactured by Thermo Scientific K.K.) as sample 4 to obtainfluorescent dye-containing particles for antibody binding.

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized as sample 4 with the exceptions describedabove.

(Sample 3-5: Fluorescent Dye-Containing Particles)

Texas Red dye (manufactured, by Sigma-Aldrich Co.) was used as afluorescent dye.

A phenol resin was used for the particles for containing the dye.

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized as sample 4 with the exceptions describedabove.

(Sample 5-6: Fluorescent Dye-Containing Particles)

Texas Red dye (manufactured by Sigma-Aldrich Co.) was used as afluorescent dye.

Polyfuran was used for the particles for containing the dye.

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized as sample 4 with the exceptions describedabove.

(Sample 5-7: Fluorescent Dye-Containing Particles)

Texas Red dye (manufactured by Sigma-Aldrich Co.) was used as afluorescent dye.

A PS/GMA complex was used for the particles for containing the dye.

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized as sample 4 with the exceptions describedabove.

(Sample 5-8: Fluorescent Dye-Containing Particles)

Texas Red dye (manufactured by Sigma-Aldrich Co.) was used as afluorescent dye.

A PS/PMMA complex was used for the particles for containing the dye.

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized as sample 4 with the exceptions describedabove.

(Sample 5-9: Fluorescent Dye-Containing Particles)

Texas Red dye (manufactured by Sigma-Aldrich Co.) was used as afluorescent dye.

A PS/acrylonitrile complex was used for the particles for containing thedye.

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized as sample 4 with the exceptions describedabove.

(Sample 5-10: Fluorescent Dye-Containing Particles)

Texas Red dye (manufactured by Sigma-Aldrich Co.) was used as afluorescent dye.

A PS/acetonitrile/GMA complex was used for the particles for containingthe dye.

Fluorescent dye-containing particles to which the anti-human antibodieswere bound were synthesized as sample 4 with the exceptions describedabove.

(Sample 6: Fluorescent Nanoparticle-Containing Particles)

As fluorescent nanoparticles, 10 μL of a CdSe/ZnS decane dispersion(Qdot655, Invitrogen Corporation) was provided, and the dispersion wasmixed with 40 μL of tetraethoxysilane. Meanwhile, 4 mL of ethanol and 1mL of 14% aqueous ammonia were mixed, followed by stirring at roomtemperature. To this mixture, the above mixture of the CdSe/ZnS decanedispersion and tetraethoxysilane prepared above was added, followed bystirring for 12 hours after the addition, to obtain fluorescentnanoparticle-containing particles. The reaction solution was centrifugedat 10000 G for 30 minutes, and the supernatant was removed. Thereafter,ethanol was added to disperse the precipitate therein, and the solutionwas centrifuged again for washing the fluorescentnanoparticle-containing particles. The particles were washed once witheach of ethanol and pure water by the same procedure. The obtainedfluorescent nanoparticle-containing particles were observed with an SEM.The average particle diameter was 120 nm, and the coefficient wasvariation of 12%.

The obtained fluorescent nanoparticle-containing particles wereadjusted, to a 3 nM concentration with a phosphate buffer physiologicalsaline solution (PBS) containing 2 mM ethylenediaminetetraacetic acid(EDTA). This solution was mixed with SM(PEG) 12(succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol] ester,manufactured by Thermo Scientific K.K.) in a final concentration of 10mM, followed by reaction for 1 hour. The mixture was centrifuged at10000 G for 20 minutes. The supernatant was removed, and the precipitatewas dispersed in PBS containing 2 mM EDTA. The solution was centrifugedagain. The precipitate was washed three times by the same procedure toobtain fluorescent nanoparticle-containing particles for antibodybinding.

Meanwhile, anti-human ER antibodies were subjected to reductiontreatment with 1M dithiothreitol (DTT), and excess DTT was removed by agel filtration column to obtain a solution of the reduced antibodiescapable of bonding to fluorescent nanoparticle-containing particles.

The fluorescent nanoparticle-containing particles for antibody bindingand the reduced antibodies prepared above were mixed in PBS containing 2mM EDTA, followed by reaction for 1 hour. The reaction was stopped, byadding 1 mM mercaptoethanol to the reaction system. The obtainedsolution was centrifuged at 10000 G for 20 minutes, and the supernatantwas removed. Thereafter, PBS containing 2 mM EDTA was added to dispersethe precipitate therein. The solution was centrifuged again. Theprecipitate was washed three times by the same procedure to obtainfluorescent nanoparticle-containing particles to which the anti-human ERantibodies were bound.

(Sample 7: Fluorescent Nanoparticle-Containing Particles)

The same fluorescent nanoparticle-containing particles to which theanti-human ER antibodies were bound as sample 6 were prepared.

In the observation described below, sample 6 was observed at anexcitation wavelength of 375 nm, and sample 7 was observed at anexcitation wavelength of 575 nm. That is, the same fluorescentnanoparticles prepared as samples 6 and 7 were observed at differentexcitation wavelengths.

(Sample 8: Fluorescent Nanoparticle-Containing Particles)

A CdSe/ZnS decane dispersion (Qdot605, Invitrogen Corporation) was usedas fluorescent nanoparticles.

Fluorescent nanoparticle-containing particles to which the anti-humanantibodies were bound were prepared as sample 6 with the exceptiondescribed above.

(Sample 9: Fluorescent Nanoparticle-Containing Particles)

A CdSe/ZnS decane dispersion (Qdot705, Invitrogen Corporation) was usedas fluorescent nanoparticles.

Fluorescent nanoparticle-containing particles to which the anti-human ERantibodies were bound were prepared as sample 6 with the exceptiondescribed above.

(Sample 11: Fluorescent Dye)

A fluorescent dye was prepared by binding CIS dye (manufactured byInvitrogen Corporation) to anti-human ER antibodies as sample 1.

(Sample 12: Fluorescent Dye)

A fluorescent dye was prepared, by binding TAMRA dye (manufactured byPCC) to anti-human ER antibodies by the same way as sample 1.

(Sample 13: Fluorescent Dye)

A fluorescent dye was prepared by binding FITC dye (manufactured by PCC)to anti-human ER antibodies by the same way as sample 1.

(Sample 14: Fluorescent Nanoparticles)

The same fluorescent, nanoparticles to which the anti-human ERantibodies were bound as sample 1 were prepared.

Sample 14 was excited at an excitation wavelength of 300 nm in theobservation described below,

(Sample 15: Fluorescent Nanoparticles)

The same fluorescent nanoparticles to which, the anti-human ERantibodies were bound as sample 1 were prepared.

Sample 15 was excited at an excitation wavelength of 500 nm in theobservation described below,

(Sample 16: Fluorescent Nanoparticles)

CdSe/ZnS fluorescent nanoparticles (Qdot565, Invitrogen Corporation)modified, with PEG having amino groups at the ends were used, asfluorescent nanoparticles for antibody binding.

Fluorescent nanoparticles to which the anti-human ER antibodies werebound were prepared, as sample 1 with the exception described above.

(Sample 17: Fluorescent Dye-Containing Particles)

FITC dye (manufactured by PCC) was used as a fluorescent dye.

Fluorescent dye-containing particles to which the anti-human ERantibodies were bound were synthesized as sample 4 with the exceptiondescribed above.

(Sample 18: Fluorescent Nanoparticle-Containing Particles)

The same fluorescent nanoparticle-containing particles to which theanti-human ER antibodies were bound as sample 6 were prepared.

Sample 18 was excited at an excitation wavelength of 300 nm in theobservation described below.

(Sample 19: Fluorescent Nanoparticle-Containing Particles)

The same fluorescent nanoparticle-containing particles to which theanti-human ER antibodies were bound as sample 6 were prepared.

Sample 19 was excited at an excitation wavelength of 500 nm in theobservation described below.

(Sample 20: Fluorescent Nanoparticle-Containing Particles)

A CdSe/ZnS decane dispersion (Qdot565, Invitrogen Corporation) was usedas fluorescent nanoparticles.

Fluorescent nanoparticle-containing particles to which the anti-human ERantibodies were bound were prepared as sample 6 with the exceptiondescribed above.

[Evaluation by Tissue Staining]

Human mammary tissue was subjected to immunostaining and staining formorphological observation (HE staining) with samples 1 to 9 and 11 to20.

A tissue array slide (CB-A712) manufactured by Cosmo Bio Co. Ltd wasused as a section to be stained. The tissue array slide wasdeparaffinized and then subjected to displacement washing with water andautoclave treatment in a 10 mM citrate buffer solution (pH 6.0) for 15minutes to activate the antigen. The tissue array slide after theactivation treatment of the antigen was washed with a PBS buffersolution and was subjected to blocking treatment with a PBS buffersolution containing 1% BSA in a moist chamber for 1 hour. After theblocking treatment, each of samples 1 to 9 and 11 to 20 diluted with aPBS buffer solution containing 1% BSA to a 0.05 nM concentration wasreacted with the tissue section for 3 hours. After the reaction withsample 1 to 9 and 11 to 20, the tissue array slide was washed with a PBSbuffer solution.

After the immunostaining, staining for morphological observation (HEstaining) was performed.

The immunostained section was stained with hematoxylin by Mayer'shematoxylin solution for 5 minutes, and then washed with running water(about 45° C.) for 3 minutes. Subsequently, the section was stained witheosin by a 1% eosin solution for 5 minutes and was immersed in pureethanol for 5 minutes. The immersion in pure ethanol was repeated fourtimes for washing and dehydration. Subsequently, the section wasimmersed, in xylene for 5 minutes four times for clearing. Finally,sealing with a sealing agent, Entellan New (manufactured by Merck KGaA),was performed to obtain a sample slide for observation.

The tissue sections which were immunostained with samples 1 to 9 and 11to 20 and subjected to staining for morphological observation wereirradiated with excitation light to emit fluorescence, and images fromthe tissue sections were obtained with an inverted fluorescencemicroscope (manufactured by Carl Zeiss).

The excitation wavelength (nm) and the fluorescence wavelength (nm) wereset with an optical filter (the excitation wavelengths and thefluorescence wavelengths of optical filters shown in Tables 1 to 4 arecentral values). The exposure conditions for obtaining microscopicimages at each excitation wavelength were adjusted so that the totalirradiation energy approximately at the focal point was 50 J.

The brightness of each pixel was calculated from the obtained image withImage-J, and the average brightness (labeled site brightness) of thesites stained with the fluorescent label (labeled site) was calculated.The average brightness corresponds to a signal value (S). A brightnessof “0” is black (most dark), and a brightness of “255” is white (mostbright). In addition, the average brightness (eosin-stained sitebrightness) of the sites that were not labeled with the fluorescent dye,but stained with eosin in the area near the fluorescent labeled cells(eosin-stained site) was calculated. The average brightness correspondsto a noise value (N).

A ratio of the labeled site brightness to the eosin-stained sitebrightness was determined as an S/N ratio. An S/N ratio of 1.5 or moreprovided easy discrimination of a labeled site from an eosin-stainedsite. Accordingly, the S/N ratio of 1.5 was determined as a criterionvalue.

Fluorescence was also visually observed during the obtainment ofmicroscopic images to evaluate the availability of visual observation.

In Tables 1 to 4, “Experimental Examples 1 to 9 and 11 to 20”corresponding to the evaluation experiments with samples 1 to 9 and 11to 20 are shown. Tables 1 to 4 show the type of fluorescent label, theexcitation wavelength, the fluorescence wavelength, the labeled sitebrightness, the eosin-stained site brightness, the S/N ratio, thediscriminateness (S/N ratio evaluation), the color visibility of thefluorescent label (visual observability) of each of ExperimentalExamples 1 to 9 and 11 to 20.

At the S/N ratio of 1.5 or more, a labeled site can be easilydiscriminated from an eosin-stained site. Accordingly, as for thediscriminateness (S/N ratio evaluation), a ratio of 1.5 or more isdetermined to be “O (suitable;”, and a ratio of less than 1.5 isdetermined to be “X (unsuitable)”.

As for the color visibility of the fluorescent label (visualobservability), a fluorescent label that was easily recognized, byvisual, observation was determined to be “O”, a fluorescent label thatwas difficult to be recognized is determined to be “Δ”, and afluorescent label that was not recognized at all is determined to be“X”.

TABLE 1 EXPERIMENTAL EXAMPLE 1 2 3 4 (Example) (Example) (Example)(Example) Sample 1 2 3 4 Fluorescent label Fluorescent nanoparticleFluorescent dye- containing particle Excitation 375 575 575 600wavelength (nm) Fluorescence 640 640 605 640 wavelength (nm) Brightnessof 21.0 10.5 10.5 105.0 labeled site Brightness of eosin- 14.0 7.0 7.25.6 stained site S/N ratio 1.5 1.5 1.5 18.8 Discriminateness ◯ ◯ ◯ ◯(S/N ratio evaluation) Color visibility of Δ Δ ◯ Δ fluorescent label

TABLE 2 EXPERIMENTAL EXAMPLE 5-1 5-2 5-3 5-4 5-5 (Example) (Example)(Example) (Example) (Example) Sample 5-1 5-2 5-3 5-4 5-5 FluorescentFluorescent dye-containing particle label Excitation 550 585 600 600 585wavelength (nm) Fluorescence 575 620 640 640 620 wavelength (nm)Brightness of 105.0 150.0 25.0 75.0 105.0 labeled site Brightness of10.5 10.5 10.5 10.5 10.5 eosin-stained site S/N ratio 10.0 14.3 2.4 7.110.0 Discrimi- ◯ ◯ ◯ ◯ ◯ nateness (S/N ratio evaluation) Color ◯ ◯ Δ Δ ◯visibility of fluorescent label EXPERIMENTAL EXAMPLE 5-6 5-7 5-8 5-95-10 (Example) (Example) (Example) (Example) (Example) Sample 5-6 5-75-8 5-9 5-10 Fluorescent Fluorescent dye-containing particle labelExcitation 585 585 585 585 585 wavelength (nm) Fluorescence 620 620 620620 620 wavelength (nm) Brightness of 90.0 85.0 102.0 110.0 80.0 labeledsite Brightness of 10.5 10.5 10.5 10.5 10.5 eosin-stained site S/N ratio8.6 8.1 9.7 10.5 7.6 Discrimi- ◯ ◯ ◯ ◯ ◯ nateness (S/N ratio evaluation)Color ◯ ◯ ◯ ◯ ◯ visibility of fluorescent label

TABLE 3 EXPERIMENTAL EXAMPLE 6 7 8 9 (Example) (Example) (Example)(Example) Sample 6 7 8 9 Fluorescent label Fluorescentnanoparticle-containing particle Excitation wavelength 375 575 575 375(nm) Fluorescence wavelength 640 640 605 720 (nm) Brightness of labeled210.0 105.0 105.0 84 site Brightness of eosin- 14.0 7.0 7.2 5.6 stainedsite S/N ratio 15.0 15.0 14.6 15 Discriminateness ◯ ◯ ◯ ◯ (S/N ratioevaluation) Color visibility of ◯ ◯ ◯ X fluorescent label

TABLE 4 EXPERIMENTAL EXAMPLE 11 12 13 14 15 16 (Comparative (Comparative(Comparative (Comparative (Comparative (Comparative Example) Example)Example) Example) Example) Example) Sample 11 12 13 14 15 16 Fluorescentlabel Fluorescent dye alone Fluorescent nanoparticle Excitationwavelength 600 550 470 300 500 475 (nm) Fluorescence wavelength 640 575500 640 640 565 (nm) Brightness of labeled 5.7 10.6 35.7 15.4 14.7 24.5site Brightness of eosin- 5.6 10.5 35.0 14.0 13.3 21.0 stained site S/Nratio 1.0 1.0 1.0 1.1 1.1 1.2 Discriminateness x x x x x x (S/N ratioevaluation) Color visibility of Δ ∘ ∘ Δ Δ ∘ fluorescent labelEXPERIMENTAL EXAMPLE 17 18 19 20 (Comparative (Comparative (Comparative(Comparative Example) Example) Example) Example) Sample 17 18 19 20Fluorescent label Fluorescent dye- Fluorescent nanoparticle- containingparticle containing particle Excitation wavelength 470 300 500 475 (nm)Fluorescence wavelength 500 640 640 565 (nm) Brightness of labeled 36.4123.2 117.6 171.5 site Brightness of eosin- 35.0 98.0 93.1 136.5 stainedsite S/N ratio 1.0 1.3 1.3 1.3 Discriminateness x x x x (S/N ratioevaluation) Color visibility of ∘ Δ Δ ∘ fluorescent label

As shown in Tables 1 to 4, Experimental Examples 1 to 9 and 11 to 13compare the cases of using fluorescent nanoparticles, fluorescentdye-containing particles, or fluorescent nanoparticles-containingparticles with the oases of using fluorescent dye alone, in the cases ofusing fluorescent nanoparticles, fluorescent dye-containing particles,and fluorescent nanoparticles-containing particles, the difference inbrightness between the labeled sites and the eosin-stained sites waslarger, and the S/N ratios of the fluorescent labels were higher,compared to the cases of the fluorescent dye alone. In the cases ofusing the fluorescent dye alone, S/N ratios were low so that visualobservation of the labeled sites was impossible.

Experimental Examples 1 to 3 and 14 to 16 compare the oases of excitingfluorescent nanoparticles at the excitation wavelengths different fromthat of eosin (Experimental Examples 1 to 3) with the cases of excitingat excitation wavelengths coinciding with the excitation wavelength ofeosin (Experimental Examples 14 to 16). The S/N ratios were higher whenthe excitation wavelengths were different from the excitation wavelengthof eosin than the S/N ratios when the excitation wavelengths coincidedwith the excitation wavelength of eosin. Similar relationships are alsoobtained in Experimental Examples 4, 5, and 17 using fluorescentdye-containing particles and Experimental Examples 6 to 9 and 18 to 20using fluorescent nanoparticles-containing particles.

As described above, a high S/N ratio and discrimination betweenimmunostaining and staining for morphological observation can beachieved in the case of using a fluorescent label of specificfluorescent nanoparticles, fluorescent dye-containing particles, orfluorescent nanoparticles-containing particles having an excitationwavelength in a wavelength region (a wavelength region of 350 to 450 nmor a longer wavelength region of 550 nm or more) other than theexcitation wavelength regions (a range of less than 350 nm and a rangeof higher than 450 nm and less than 550 nm) of eosin.

Experimental Examples 7 to 9 compare the emission color visibilities ofthe fluorescent label at different fluorescence wavelengths. InExperimental Example 9, the emission wavelength (fluorescencewavelength) was close to the near-infrared region, and the labeled sitewas therefore difficult to be recognized by visual observation. In orderto confirm a labeled site by visual observation, the fluorescent labelpreferably has an emission wavelength of 550 to 700 nm.

INDUSTRIAL APPLICABILITY

The present invention can be used for specifically detecting abiological substance in a pathological section when staining formorphological observation and immunostaining are simultaneouslyperformed.

The invention claimed is:
 1. A biological substance detection method ofspecifically detecting a biological substance in a pathological section,the method comprising: immunostaining the pathological section with afluorescent label; staining the pathological section with eosin used asa staining reagent for morphological observation; and detecting thebiological substance in the pathological section, which ismorphologically stained with the eosin, through fluorescence emission ofthe fluorescent label by irradiation of the stained pathological sectionwith excitation light having a wavelength of 350 to 450 nm or 550 nm ormore, wherein in the immunostaining of the pathological section, afluorescent dye-containing particle comprising an organic or inorganicmaterial particle containing a plurality of fluorescent dye moleculesand having a peak of excitation spectrum at a wavelength region of 350to 450 nm or 550 nm or more is used as the fluorescent label, and thefluorescent dye-containing particle binds to the biological substancevia an antibody against the biological substance.
 2. The biologicalsubstance detection method according to claim 1, wherein the particlefor containing fluorescent dye of the fluorescent dye-containingparticle is composed of one or more material selected from polystyrene,polyamide, polylactic acid, polyacrylonitrile, polyglycidylmethacrylate, polymelamine, polyurea, polybenzoguanamine, polyfuran,polyxylene, phenol resins, and polysaccharides.
 3. The biologicalsubstance detection method according to claim 1, wherein the fluorescentdye of the fluorescent dye-containing particle is composed of one ormore material selected from rhodamine dye molecules,boron-dypyrromethene, Sulforhodamine 101, squarylium dye molecules,cyanine dye molecules, oxazine dye molecules, and carbopyronine dyemolecules.
 4. The biological substance detection method according toclaim 1, wherein in the immunostaining of the pathological section, afluorescent label having an emission wavelength in a wavelength regionof 550 to 700 nm is used as the fluorescent label.
 5. The biologicalsubstance detection method according to claim 1, wherein in theimmunostaining of the pathological section, a fluorescent label havingan emission wavelength in a wavelength region of 590 to 650 nm is usedas the fluorescent label.
 6. The biological substance detection methodaccording to claim 1, wherein in the immunostaining of the pathologicalsection, a fluorescent label having an emission wavelength in awavelength region of 590 to 630 nm the fluorescent label.
 7. Thebiological substance detection method according to claim 1, wherein thefluorescent dye-containing particle size ranges from 50 to 200 nm.